Device for enhancing immunostimulatory capabilities of T-cells

ABSTRACT

T-cells are generated with enhanced immunostimulatory capabilities for use in self therapy treatment protocols, by utilizing a biodegradable device with a biodegradable support that has one or more agents that are reactive to T-cell surface moieties. The biodegradable devices are mixed with the T-cells sufficiently so that the one or more agents cross-link with the T-cells&#39; surface moieties and deliver a signal to the T-cells to enhance immunostimulatory capabilities.

The present application is a divisional of and claims priority of U.S.patent application Ser. No. 12/687,281, filed Jan. 14, 2010, which is adivisional of and claims priority of U.S. patent application Ser. No.11/066,133, filed Feb. 24, 2005, now U.S. Pat. No. 7,678,572, issuedMar. 16, 2010, the content of which is hereby incorporated by referencein its entirety.

The present application is based on and claims the benefit of U.S.provisional patent application Ser. No. 60/547,966, filed Feb. 26, 2004,the content of which is hereby incorporated by reference in itsentirety.

FIELD OF INVENTION

This invention relates to methods for generating T-cells with enhancedimmunostimulatory capabilities for use in cell therapy treatmentprotocols.

BACKGROUND OF THE INVENTION

Cell therapy methods have been developed in order to enhance the hostimmune response to tumors, viruses and bacterial pathogens. Cell therapymethods often involve the ex-vivo activation and expansion of T-cells.Examples of these type of treatments include the use tumor infiltratinglymphocyte (TIL) cells (see U.S. Pat. No. 5,126,132 issued toRosenberg), cytotoxic T-cells (see U.S. Pat. No. 6,255,073 issued toCai, et al.; and U.S. Pat. No. 5,846,827 issued to Celis, et al.),expanded tumor draining lymph node cells (see U.S. Pat. No. 6,251,385issued to Terman), and various other lymphocyte preparations (see U.S.Pat. No. 6,194,207 issued to Bell, et al.; U.S. Pat. No. 5,443,983issued to Ochoa, et al.; U.S. Pat. No. 6,040,177 issued to Riddell, etal.; U.S. Pat. No. 5,766,920 issued to Babbitt, et al.).

For maximum effectiveness of T-cells in cell therapy protocols, the exvivo activated T-cell population should be in a state that can maximallyorchestrate an immune response to cancer, infectious diseases, or otherdisease states. For an effective T-cell response, the T-cells first mustbe activated. For activation, at least two signals are required to bedelivered to the T-cells. The first signal is normally delivered throughthe T-cell receptor (TCR) on the T-cell surface. The TCR first signal isnormally triggered upon interaction of the TCR with peptide antigensexpressed in conjunction with an MHC complex on the surface of anantigen-presenting cell (APC). The second signal is normally deliveredthrough co-stimulatory receptors on the surface of T-cells.Co-stimulatory receptors are generally triggered by correspondingligands or cytokines expressed on the surface of APCs.

Due to the difficulty in maintaining large numbers of natural APC incultures of T-cells being prepared for use in cell therapy protocols,alternative methods have been sought for ex-vivo activation of T-cells.One method is to by-pass the need for the peptide-MHC complex on naturalAPCs by instead stimulating the TCR (first signal) with polyclonalactivators, such as immobilized or cross-linked anti-CD3 or anti-CD2monoclonal antibodies (mAbs) or superantigens. The most investigatedco-stimulatory agent (second signal) used in conjunction with anti-CD3or anti-CD2 mAbs has been the use of immobilized or soluble anti-CD28mAbs.

The combination of anti-CD3 mAb (first signal) and anti-CD28 mAb (secondsignal) immobilized on a solid support such as paramagnetic beads (seeU.S. Pat. No. 6,352,694 issued to June, et al.) has been used tosubstitute for natural APCs in inducing ex-vivo T-cell activation incell therapy protocols (Levine, Bernstein et al. 1997; Garlic, LeFeveret al. 1999; Shibuya, Wei et al. 2000). While these methods are capableof achieving therapeutically useful T cell populations, the use ofparamagnetic beads makes the ease of preparation of T-cells less thanideal. Problems include the high cost of the beads, the labor-intensiveprocess for removing the beads prior to cell infusion, and the inabilityof the beads to activate CD8 T-cell subsets (Deeths, Kedl et al. 1999;Laux, Khoshnan et al. 2000). In addition, the T-cell populationsresulting from this method, and other prior art T-cell stimulationmethods, lack the type of robustness required for eliciting effectiveimmune stimulation when infused into patients. As a consequence, noprior art cell therapy protocols have demonstrated significant efficacyin clinical settings.

This has motivated the search for more effective methods for activatingT-cells for use in cell therapy protocols. One such method is the use ofAPC tumor cell lines that have been genetically modified to expressreceptors that bind mAbs. These modified APC can be loaded with anti-CD3and anti-CD28 mAbs (Thomas, Maus et al. 2002) or additionally modifiedto express the ligand for 4-1BB (Maus, Thomas et al. 2002) and then usedto activate T-cells for use in cell therapy protocols. It was found thatthese modified APCs resulted in more effective activation of T-cellpopulations than the use of CD3/CD28-coated paramagnetic beads. However,the use of genetically-manipulated tumor cell lines in cell therapyprotocols raises safety concerns which limit the commercial applicationof this technique.

SUMMARY OF THE INVENTION

In this situation, biodegradable supports coated with a first materialthat is capable of cross-linking second materials with reactivity tomoieties on the surface of T-cells are utilized. The coatedbiodegradable supports are then mixed with second material labeledT-cells. The signals delivered by the cross-linked second materials areenhanced by centrifugation of the mixture. The signals are furtherenhanced by the culture of the mixture at high cell densities.

The present invention also includes biodegradable devices that have abiodegradable support with one or more agents that are reactive toT-cell moieties. Such agents deliver signals to T-cells to enhanceimmunostimulatory or immunoregulatory capabilities.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

There is a need for improved T-cell stimulation methods capable ofincreasing the robustness of T-cells for use in cell therapy protocolsthat are more suitable for use in human therapy.

In order to improve the robustness of T-cells, it is also desirable thatthe improved stimulation methods as closely as possible mimic thestimulatory effects of natural APCs. The improvement in T-cellactivation observed with the CD3/CD28-coated APC cell lines discussedabove (Thomas, Maus et al. 2002); (Maus, Thomas et al. 2002), wasattributed to the availability of ligands to co-stimulatory moleculesnaturally expressed on the APC cell line that worked in concert with theCD3/CD28 stimulation. These ligands included B7-H3, PD-L1, PD-L2 andIL-15.

Therefore, it is desired to have a method for improved T-cellstimulation capable of presenting a multiplicity of co-stimulatoryligands without the requirement for use of a tumor cell line.

Natural APCs, however, not only provide multiple simultaneous stimuli toT-cells, they provide different arrays of multiple stimuli at differenttimes and/or stages in the T-cell response to T-cell stimulation. Noprior art T-cell stimulation methods are capable of mimicking thisnatural process.

The ability to mimic this natural process would provide a means tocontrol not only the expansion of T-cells, but also the differentiationof T-cells. In the process of T-cell differentiation into regulatory oreffector cells, different signals are required at different times and/orstages in the T-cell response to APC stimulation. Thus, it would bedesirable to be able to create ex-vivo conditions that mimic thisnatural process in order to provide a greater variety of differentiatedcells for use in cell therapy, including cells which could eitherstimulate immunity or suppress immunity.

The maintenance of the high density cell cultures used in the presentinvention require special care, as the degradation of the biologicalsupports causes a fall in the media pH and the higher cell densitiesresult in rapid accumulation of metabolic waste products and consumptionof nutrients in the culture medium. For these reasons, media changes arerequired at least daily and preferably at least twice daily after thecells obtain a cell density in excess of 1 million per ml.

Frequent media changes can remove endogenous cytokines that areimportant for the maintenance and growth of the T-cell cultures.Therefore, in preferred embodiments, the removed culture media isfiltered through a dialysis membrane in order to remove metabolic wasteproducts, but retain endogenous cytokines. The retained media is thensupplemented with fresh nutrient media and returned to the mixedculture. This enables the cells to be exposed to fresh nutrient mediawithout dilution of the endogenous cytokines.

As the T-cells grow and mature in the cultures, various arrays of secondmaterials can be added to the cultures at any time as required andsubsequently cross-linked by mixing with additional coated biodegradablesupports. Alternatively, the second materials can be added to thebiodegradable supports and the coated supports added at various times tothe cultures. Centrifugation of the mixture each time after addingadditional second materials and coated biodegradable supports providesadded benefit. In preferred embodiments, the centrifugation step isconducted daily to coincide with the media dialysis step.

Biodegradable Spheres

Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolicacid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone)(PCL), and polyanhydrides are preferred materials for use asbiodegradable polymers for the supports. The polymers can be formulatedas various shapes, such as films, strips, fibers, gels, nanospheres ormicrospheres, and then coated with a first material. Microspheres are apreferred formulation because they can be reproducibly manufactured intosmall microsphere particle sizes of 1.to.500 microns, preferably 1 to 10microns and most preferably 1 to 5 microns. Microspheres of this sizerange are capable of direct injection into the body by conventionalmethods. It is preferred that the coated microspheres be formulated todegrade in culture media or physiological fluids within 14 days, morepreferably within 7 days, and most preferably within 3 days. In otherpreferred methods, nanospheres are formulated. These devices arepreferred in applications where very rapid degradation, for example 3days or less is required.

One preferred first material for coating on the biodegradablemicrospheres is polyclonal goat (or sheep) anti-mouse polyclonalantibodies. By way of example, this preferred first material can be usedto cross-link mouse-derived monoclonal antibodies, or fragments orgenetically engineered derivatives thereof, that have specificity forT-cell surface moieties. Thus, for example, the mixing of goatanti-mouse coated microspheres (or nanospheres) with human T-cellslabeled with mouse anti-human CD3 and mouse anti-human CD28 mAbs willcause the cross-linking of the mouse mAbs on the human T-cells throughthe binding of the goat anti-mouse polyclonal antibody with the mousemAbs. The cross-linking of the mAbs causes the activation andproliferation of the T-cells. Many combinations of first materials andsecond materials can be used to accomplish the objective ofcross-linking second agents attached to T-cell surface moieties in orderto initiate signal transduction and activation of T-cells.Alternatively, the second materials can be added to the biodegradablesupports prior to addition to the T-cells.

The coated biodegradable microspheres (or nanospheres) used in thepresent invention provide many advantages for preparation of T-cells foruse in cell therapy protocols over prior art methods where mitogenicagents are immobilized on a solid surface, such as paramagnetic beads:

First, since the devices are biocompatible and naturally degrade intonon-toxic substances, there is no need to institute a bead removalprocess.

Second, because the devices have a low density, they can be used withcells being subjected to a centrifugal force. Prior art devices, such asparamagnetic beads, cause damage to cells when subjected tocentrifugation. The ability to centrifuge cells with the beads permitsthe use of centrifugal force to enhance the quality of signals providedto the T-cells by stimulatory ligands cross-linked on the surface of theT-cells and also provides a means to wash and otherwise process theT-cells for preparation for infusion.

Third, in one use of the present invention, rather than immobilizingT-cell stimulatory and co-stimulatory ligands to a solid surface topresent signals to T-cells, the use of a coated biodegradablemicrospheres (or nanospheres) permits the ligands to be first applied tothe T-cells and then the labeled T-cells to be mixed with the coatedbiodegradable microspheres (or nanospheres). In this manner, the coatedmicrospheres (or nanospheres) act as a universal cross-linking agent.

Fourth, as a universal cross-linking agent, a multiplicity ofstimulatory and co-stimulatory ligands can be applied to T-cells and becross-linked by the coated beads and the composition of the multiplicityof stimulatory and co-stimulatory ligands to be cross-linked can bevaried over time.

Fifth, the ability to vary the composition of the array of stimulatoryand co-stimulatory signals provided to T-cells over time permits thepractice of methods designed to mimic natural presentation of T-cellproliferation, differentiation and functional signals.

Sixth, the ability to mimic the natural signal presentation to T-cellspermits the development of T-cells with a multitude of functionalcharacteristics for use in cell therapy protocols.

Seventh, the ability to control the sequence and variety of signalsdelivered to T-cells over time permits a means to control thedifferentiation pathways of T-cells ex-vivo. This will permitexperimentation with novel combinations and sequencing of signalsdelivered to T-cells. Such methods will lead to T-cell products withnovel effector functions both stimulatory and suppressive for use incell therapy protocols.

For the purposes of the present invention, all references to T-cellsincludes a population of cells with at least a portion of the cellscontaining T-cells. T-cells are cells which express TCR, including α/βand γ/δ TCRs. T-cells include all cells which express CD3, includingT-cell subsets which also express CD4 and CD8. T-cells include bothnaïve and memory cells and effector cells such as CTL. T-cells alsoinclude regulatory cells such as Th1, Tc1, Th2, Tc2, Th3, Treg, and Tr1cells. T-cells also include NKT-cells and similar unique classes of theT-cell lineage.

Increased Signal Transduction

One aspect the present invention provides methods for enhancedstimulation of a population of T-cells by the concentration of a mixtureof first material coated biodegradable microspheres (or nanospheres) andsecond material labeled T-cells. In order to increase the efficacy ofthe signal transduced to the T-cells, it is important to both increasethe quantity of second agents cross-linked and the quality of thecross-linking.

In order to assure the highest quantity of second materials that areassociated with the corresponding surface moieties on the surface of theT-cells, the labeling of the T-cells should be conducted with excesssecond materials. In a preferred embodiment where mouse mAbs to humanT-cell surface antigens are the second materials, the mAbs arepreferably mixed with a T-cell suspension whereby the T-cells are at aconcentration of 1×10⁶ to 1×10⁷ per ml and each mAb is at aconcentration of 0.5 μl/ml to 10 μl/ml, preferably 1 μl/ml. The labeledT-cells should be mixed with the coated biodegradable spheres at a ratioof at least one sphere per cell, and preferably at a ratio of 3 spheresper cell.

In order to assure the highest quality of cross-linking, the labeledcells and the coated biodegradable spheres are preferably first mixedthoroughly and then concentrated together under centrifugal force. Thecentrifugation is preferably conducted every 3 days, more preferably atleast once daily. It is also preferable that the T-cells be kept at 4°C. from the time new mAbs are added through the completion of thecentrifugation. Keeping the cells at refrigeration temperature preventsthe capping and shedding of the ligated T-cell surface receptors priorto being cross-linked.

Cell Culture Methods

It is preferable to maintain processive and sustained TCR signaltransduction and co-simulation in order to provide the most robustT-cells for use in cell therapy protocols. For this reason, the methodsof the present invention work best when the cultured T-cells aremaintained at high cell densities, such as greater than 10⁶ cells/ml, ormore preferably greater than 10⁷ cells/ml, or most preferably greaterthan 10⁸ cells/ml. The high cell densities increase the cell:cellinteraction and the interaction with the biodegradable spheres.

The increased cell:cell interaction has a beneficial effect that isseparate from the cross-linking effect of the biodegradable spheres. Thebeneficial effect comes from the expression of stimulatory ligands whichupregulate on the surface of T-cells in response to maximal activationconditions. These ligands interact with the corresponding receptors onother T-cells. For example, T-cells will express one or more of thefollowing TNFR co-stimulatory ligands such as LIGHT, CD70, OX40L, 4-1BBLand CD30L after maximal activation.

Maintaining cells at high densities in culture with biodegradablespheres requires the frequent changing of the culture media. The highcell densities result in a high rate of build up of metabolic wasteproducts and consumption of available nutrients. In addition, thehydrolysis of the biodegradable spheres causes the pH of the culturemedia to become acidic. Too rapid media replacement, however, can bedetrimental to cultures where exogenous cytokines are not utilized. Itis preferable not to use exogenous cytokines when processing cells foruse in cell therapy protocols, as exogenous cytokines can be toxic wheninfused into humans and can make the cultured cells dependant upon thepresence of the exogenous cytokines for viability. Therefore, themethods of the present invention include a dialysis step in the cellprocessing.

Dialysis of the culture medium with membrane pore size of 10,000 daltonor less will enable retention of endogenous cytokines while allowingpassage of metabolic waste. In preferred embodiments, half the culturemedium of a culture is removed daily and 90% passed through a dialysisfilter. The media passed through the filter is discarded, while theretained media is brought up to the original volume with fresh culturemedia.

According to the method of the present invention, a process is describedfor producing T-cells with robustness and enhanced function for use incell therapy protocols involving: (1) the labeling of a population ofT-cells with one or more agents that have reactivity to cell surfacemoieties; (2) mixing of the population of labeled T-cells with coatedbiodegradable spheres capable of cross-linking the agents attached tocell surface moieties on the T-cells causing a signal to be transducedto the T-cells; (3) concentrating of the mixture by centrifugation; (4)continued culture of the T-cells at high cell density; and (5) removalof media from the cultures at least daily and the dialysis of the mediafor retention of endogenous cytokines and replacement with fresh media;and (6) repeat of the process as necessary with the same or differentagents for labeling of the T-cells in order to generate both thequantities of T-cells necessary for infusion and the optimal function ofthe T-cells for clinical effect.

Choice of T-Cell Ligating Targets

The ability to design more efficient and effective T-cell activation,expansion and differentiation methods will be a direct result of theselection and timing of application of second materials. Secondmaterials are agents which are capable of ligating T-cell surfacemoieties and delivering a signal to the T-cell upon cross-linking. Thesematerials are preferably monoclonal antibodies, or fractions orgenetically manipulated versions thereof, such as fusion proteins. Theselection of second materials will be as a result of understanding ofthe T-cell activation, expansion and differentiation process and therequirements for the type and duration of signals at any one time in thelife of the responding T-cells.

It is known that at least two type of receptors need to be engaged forT-cell activation, the TCR and a co-stimulator (Chambers and Allison1999). In response to natural APC engagement with antigenic peptide andco-stimulatory ligands, the contact site of the APC and T-cell forms an“immunological synapse”. The synapse assembles into topologically andspatially distinct regions. The initial TCR engagement occurs at theperiphery of the synapse (Grakoui, Bromley et al. 1999) after whichligand engagement of co-simulating molecules such as CD28, CD2, CD48 andLFA-1 facilitates the sorting and re-arrangements of receptors at thesynapse. The content of molecules at the synapse can be specificallyenriched in a subset of proteins and can selectively exclude proteins.This selective movement of proteins is facilitated by structures knownas “lipid rafts”.

Lipid raft membrane partitioning is known to be crucial for optimal TCRsignal transduction (Moran and Miceli 1998; Janes, Ley et al. 1999) andco-stimulators to TCR signaling cause the synapse formation and there-organization and clustering of lipid rafts at the synapse. Theseevents provide a natural mechanism for integrating spatial and temporalinformation provided to T-cells from the environment.

Accordingly, knowledge of the types of receptors available at thesynapse in response to defined stimuli can provide the information fordeciding the various types of co-stimulators to utilize over a period oftime. Lipid rafts function as platforms for the concentration andjuxtaposition of TCR associated signal transducers and assembly of anorganized TCR signaling complex. Thus, by a process of first providing adefined array of signals to a population of T-cells and next analyzingthe proteins assembled in lipid rafts that were induced by the firstarray, a second array of possible signals can be determined. The processcan be repeated with second array stimulators. After application of thesecond array, the process can be repeated with a third array and so on.At each step in the process, the response of the T-cells can bemonitored in order to optimize for the desired function, such asproliferation, the types and quantities of selected cytokine production,the expression of effector molecules and other functional surfacemolecules.

For example, both CD2 and LFA-1 are raft associated proteins that canstimulate initial T-cell activation in the absence of CD28 engagement(Yashiro-Ohtani, Zhou et al. 2000). The engagement of these molecules isknown to upregulate and increase avidity for receptors for ICAM-1 whichcould then be engaged in a second array. CD2/LFA-1 engagement are knowto facilitate T-cell activation by increasing the number of TCRs engagedover time, whereas CD28 functions by increasing the potency of thoseTCRs that are engaged, thus lowering the number of TCRs that need to beengaged in order to effect a response (Bachmann, McKall-Faienza et al.1997).

In preferred embodiments, a first array including CD3 and otherco-stimulatory molecules selected from one or more of the following:CD2, CD28, CD48, LFA-1, CD43, CD45, CD4, CD8, CD7, GM1, LIGHT (HVEMfusion protein) is utilized. A second array including CD3 and one ormore of the first array co-stimulators with the additional choices ofthe following inducible co-stimulatory ligands: CD27, OX40, 4-1BB andCD30.

Also in preferred embodiments, T-cell counter receptors to variousadhesion molecules can be engaged during the process. Examples ofadhesion molecules on T-cells are: CD44, CD31, CD18/CD11a (LFA-1), CD29,CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA-4). Othersuitable second array agents include non-cytokine agents which bind tocytokine receptors and deliver a signal when cross-linked Examples ofthese type of agents are mAbs to cytokine receptors including: IL-2R,IL-4R, IL-10R, Type H IFNR1 and R2, Type I IFNR, IL-12Rbeta1 and beta2,IL-15R, TNFR1 and TNFR2, and IL-IR. Also any agents capable of bindingto chemokine receptors on T-cells and delivering a signal whencross-linked, including those in the C—C and C—X—C categories. Examplesof chemokine receptors associated with T-cell function include CCR1,CCR2, CCR3, CCR4, CCR5, and CXCR3

EXAMPLE METHODS

Examples of optimized processes for producing a T-cell population withenhanced ability to stimulate the immune system follow. All examplesutilize goat anti-mouse coated biodegradable microspheres and T-cellslabeled with mouse mAbs specific for T-cell surface antigens:

Example #1

Set-up (Day 0)

-   -   (1) collection of leukocytes by leukapheresis;    -   (2) purification of 10⁸ CD4+ T-cells by positive selection;    -   (3) labeling of purified CD4+ cells with anti-CD3, anti-CD28 and        anti-IL-12Rbeta2 mAbs;    -   (4) mixing the labeled cells with coated microspheres in gas        permeable bags (3:1 sphere:cell);    -   (5) suspension of the mixture at a cell density of 1×106/ml in        100 ml;    -   (6) centrifugation of the mixture at 500×g for 8 min at 4° C.;    -   (7) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 3    -   (8) remove 80 ml of culture media by syringe aspiration using a        0.45 micron filter so as not to remove any cells;    -   (9) pass 70 ml of the removed media through a dialysis filter of        6,000 dalton cut-off size;    -   (10) add 70 ml of fresh culture media to the retained 10 ml and        add back to the culture bag;    -   (11) add 100 μg each of anti-CD3, anti-CD28, anti-IL-12Rbeta2        and anti-4-1BB mAbs to the culture bag;    -   (12) mix coated microspheres at a sphere:cell ratio of 1:1;    -   (13) centrifuge mixture at 500×g for 8 min at 4° C.;    -   (14) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 4    -   (15) repeat steps 8-10        Day 5    -   (16) repeat steps 8-10        Day 6    -   (17) repeat steps 8-14    -   (18) after 12 h repeat steps 8-10        Day 7    -   (19) repeat steps 8-10    -   (20) after 12 h repeat steps 8-10        Day 8    -   (21) repeat steps 8-10    -   (22) after 12 h repeat steps 8-10        Day 9    -   (23) harvest T-cell population and formulate for infusion        Results

This method results in a population of T-cells with enhancedproliferation and production of IFN-gamma and TNF-alpha compared tocells activated with CD3/CD28-coated immunomagnetic beads alone. N=6

Fold IFN-gamma TNF-alpha IL-4 Method Expansion ng/ml ng/ml pg/ml Example#1 830 +/− 77  970 +/− 160 180 +/− 38 <20 3/28-beads + 80 +/− 20   3 +/−2.2 0.5 +/− .2 80 +/− 16 IL-2

Example #2

Set-up (Day 0)

-   -   (4) collection of leukocytes by leukapheresis;    -   (5) purification of 10⁸ CD4+ T-cells by positive selection;    -   (6) labeling of purified CD4+ cells with anti-CD3, anti-CD28        mAbs;    -   (4) mixing the labeled cells with coated microspheres in gas        permeable bags (3:1 sphere:cell);    -   (5) suspension of the mixture at a cell density of 1×106/ml in        100 ml;    -   (6) centrifugation of the mixture at 500×g for 8 min at 4° C.;    -   (7) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 3    -   (8) remove 80 ml of culture media by syringe aspiration using a        0.45 micron filter so as not to remove any cells;    -   (9) pass 70 ml of the removed media through a dialysis filter of        6,000 dalton cut-off size;    -   (15) add 70 ml of fresh culture media to the retained 10 ml and        add back to the culture bag;    -   (16) add 100 μg each of anti-CD3, anti-CD28, mAbs to the culture        bag;    -   (17) mix coated microspheres at a sphere:cell ratio of 1:1;    -   (18) centrifuge mixture at 500×g for 8 min at 4° C.;    -   (19) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 4    -   (15) repeat steps 8-10        Day 5    -   (16) repeat steps 8-10        Day 6    -   (24) repeat steps 8-14    -   (25) after 12 h repeat steps 8-10        Day 7    -   (26) repeat steps 8-10    -   (27) after 12 h repeat steps 8-10        Day 8    -   (28) repeat steps 8-10    -   (29) after 12 h repeat steps 8-10        Day 9    -   (30) harvest T-cell population and formulate for infusion        Results

This method results in a population of T-cells with enhancedproliferation and production of IFN-gamma and TNF-alpha compared tocells activated with CD3/CD28-coated immunomagnetic beads alone, as wellas enhanced expression of CD40L. N=6

Fold IFN-gamma TNF-alpha CD40L Method Expansion ng/ml ng/ml % Example #2630 +/− 77  90 +/− 16.7 8.8 +/− 1.3 78.5 +/− 10 3/28-beads + 80 +/− 20 3+/− 2.2 0.5 +/− .2   15 +/− 6 IL-2

Example #3

Set-up (Day 0)

-   -   (7) collection of leukocytes by leukapheresis;    -   (8) purification of 10⁸ CD4+ T-cells by positive selection;    -   (9) labeling of purified CD4+ cells with anti-CD3, anti-CD28 and        anti-HVEM mAbs;    -   (4) mixing the labeled cells with coated microspheres in gas        permeable bags (3:1 sphere:cell);    -   (5) suspension of the mixture at a cell density of 1×106/ml in        100 ml;    -   (6) centrifugation of the mixture at 500×g for 8 min at 4° C.;    -   (7) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 3    -   (8) remove 80 ml of culture media by syringe aspiration using a        0.45 micron filter so as not to remove any cells;    -   (9) pass 70 ml of the removed media through a dialysis filter of        6,000 dalton cut-off size;    -   (20) add 70 ml of fresh culture media to the retained 10 ml and        add back to the culture bag;    -   (21) add 100 μg each of anti-CD3, anti-CD28, anti-CD27 and        anti-4-1BB mAbs to the culture bag;    -   (22) mix coated microspheres at a sphere:cell ratio of 1:1;    -   (23) centrifuge mixture at 500×g for 8 min at 4° C.;    -   (24) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 4    -   (15) repeat steps 8-10        Day 5    -   (16) repeat steps 8-10        Day 6    -   (31) repeat steps 8-14    -   (32) after 12 h repeat steps 8-10        Day 7    -   (33) repeat steps 8-10    -   (34) after 12 h repeat steps 8-10        Day 8    -   (35) repeat steps 8-10    -   (36) after 12 h repeat steps 8-10        Day 9    -   (37) repeat steps 8-10;    -   (38) after 12 h repeat steps 8-10;    -   (39) add 100 μg each of anti-CD3, anti-CD28, and HVEM-Fc to the        culture bag;    -   (40) mix coated microspheres at a sphere:cell ratio of 1:1;    -   (41) centrifuge mixture at 500×g for 8 min at 4° C.;    -   (42) gently resuspend and culture in humidified atmosphere at        37° C. with 5% CO₂;        Day 10    -   (43) repeat steps 8-10;    -   (44) after 12 h repeat steps 8-10;        Day 11    -   (45) harvest T-cell population and formulate for infusion.        Results

This method results in a population of T-cells with enhancedproliferation and production of IFN-gamma LIGHT and FasL compared tocells activated with CD3/CD28-coated immunomagnetic beads alone. N=6

Fold IFN-gamma LIGHT FasL Method Expansion ng/ml (%) % Example #3 290+/− 21  44 +/− 6.2 38.4 +/− 3.3 61.4 +/− 10   3/28-beads + 80 +/− 20  3+/− 2.2 6.1 +/− 5    4 +/− 1.3 IL-2

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A device for enhancing immunostimulatorycapabilities of T-cells comprising: an universal crosslinking agentcomprising a support coated with a first material, wherein the firstmaterial is capable of crosslinking more than one array of secondmaterials, the second materials capable of binding moieties on thesurface of T-cells to deliver a signal to the T-cells and wherein eachsuccessive array of second materials is added at a later time in theT-cell response than the previous array of second materials and whereinthe second materials comprise anti-chemokine receptors.
 2. The device ofclaim 1 wherein each of the one or more arrays comprises one or moresecond materials.
 3. The device of claim 1 wherein each of the one ormore arrays comprises two or more second materials.
 4. The device ofclaim 1 wherein the universal crosslinking agent is capable ofcrosslinking second materials bound to the T-cell surface moieties. 5.The device of claim 1 wherein an array of second materials comprises oneor more antibodies that have specificity to a T-cell surface moiety. 6.The device of claim 1 wherein the first material is an antibody.
 7. Thedevice of claim 1 wherein the support is a biodegradable support.
 8. Thedevice of claim 1 wherein the support is a biodegradable microsphere. 9.The device of claim 1 wherein the support is biodegradable into asubstance that is nontoxic to humans.
 10. The device of claim 1 whereinthe support is a microsphere that degrades in 14 days or less.
 11. Thedevice of claim 1 wherein the anti-chemokine receptors are in the C—Cand C—X—C categories.
 12. The device of claim 11 wherein theanti-chemokine receptors are selected from CCR1, CCR2, CCR3, CCR4, CCR5and CXCR3.
 13. The device of claim 1 wherein the first materialcrosslinks at least two arrays of second materials, each arraycomprising at least two second materials and wherein the second array isadded at a later time in the T-cell response than the first array. 14.The device of claim 1 wherein the first material crosslinks more thantwo arrays of second materials, each array comprising at least twosecond materials and wherein each successive array is added at a latertime in the T-cell response than the previous array.